Bivalent rotavirus VP4∗ stimulates protective antibodies against common genotypes of human rotaviruses

Figures

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  Figure 1. Purification and characterization of VP4∗ proteins (A) SDS-PAGE analysis of VP4∗ proteins of P[4], P[6], and P[8] genotype rotaviruses. (B) Western blot analysis of VP4∗ proteins using a broadly reactive mAb 9C4. (C) Native PAGE analysis of VP4∗ proteins. (D–F) HPSEC analysis of VP4∗ proteins using G3000PWXL. (G–I) DSC analysis of VP4∗ proteins.

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  Figure 2. Dose-dependent immunogenicity study of P[8]-VP4∗ in guinea pigs and rabbits The guinea pigs and rabbits were immunized intramuscularly (IM) with VP4∗ proteins formulated in aluminum adjuvant for three times with 2-week interval. The serum samples were collected individually from the guinea pigs and rabbits before immunization and 2 weeks after each dose of immunization. (A) Antibody titers against P[8]-VP4∗ in guinea pig sera. (B) Antibody titers against P[8]-VP4∗ in rabbit sera. (C) Neutralizing antibody titers against human rotavirus of Wa in guinea pig sera. (D) Neutralizing antibody titers against human rotavirus Wa in rabbit sera. The bars represent mean and standard error of the means in each group (n = 5). ∗, ∗∗, ∗∗∗, ∗∗∗∗ represents p < 0.05, 0.005, 0.0005, 0.0001; ns represents p > 0.05.

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  Figure 3. The immunogenicity of VP4∗ in guinea pigs and rabbits The guinea pigs and rabbits were immunized intramuscularly (IM) with VP4∗ proteins formulated in aluminum adjuvant for three times with 2-week interval. The serum samples were collected individually from the guinea pigs and rabbits before immunization and 2 weeks after each dose of immunization. (A–D) (A) The antibody titers in serum of guinea pigs, (B) the antibody titers in the serum of rabbits, (C) the neutralizing antibody titers in serum of guinea pigs, (D) the neutralizing antibody titers in serum of rabbits. Rotavirus Wa (G1P[8]), DS-1 (G2P[4]), and VR-2104 (G3P[6]) were used for detecting the homotypic neutralizing antibodies stimulated by P[4]-, P[6]-, and P[8]-VP4∗ proteins, respectively. The bars represent mean and standard error of the means in each group (n = 5). ∗, ∗∗, ∗∗∗, ∗∗∗∗ represents p < 0.05, 0.005, 0.0005, 0.0001; ns represents p > 0.05.

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  Figure 4. The cross-neutralizing antibodies against different rotavirus induced by P[8]-VP4∗ in guinea pigs and rabbits The neutralizing activity of serum samples after three doses of immunization described in Figure 3 against different rotaviruses were determined. (A and B) (A) The cross-neutralizing antibodies in the serum of guinea pigs after 3 doses immunization, (B) the cross-neutralizing antibodies in the serum of rabbits after 3 doses immunization. The serum was collected to detected neutralizing antibodies against homotypic rotavirus and heterotypic with ELISpot. The bars represent mean and standard error of the means in each group (n = 5). ∗, ∗∗, ∗∗∗, ∗∗∗∗ represents p < 0.05, 0.005, 0.0005, and 0.0001, respectively; ns represents p > 0.05.

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  Figure 5. Cross-neutralizing antibodies detected by ELISpot for the serum of guinea pigs immunized with monovalent, bivalent, and trivalent vaccine (A–D) the neutralizing antibodies against different genotype rotavirus in the serum of guinea pigs immunized three doses of monovalent, bivalent, or trivalent VP4∗ proteins. The bivalent and trivalent VP4∗ formulations include 10 μg VP4∗ proteins of each P genotype. The bars represent mean and standard error of the means in each group (n = 5). ∗, ∗∗, ∗∗∗, ∗∗∗∗ represents p < 0.05, 0.005, 0.0005, and 0.0001, respectively; ns represents p > 0.05.